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Book of AbstractsGPZ Meeting of AG Cytogenetics30−31 March 2017

Chromosome biology and genome editing in the context of plant breeding

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Editor: Andreas Houben/IPK

Assistent: Regina Devrient/IPK

Photograph: Andreas Houben/IPK

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Topic

In light of the ongoing progress in the field of cytogenetics

and genome editing, the focus of the conference is “Chro-

mosome Biology and Genome engineering in the context of

plant breeding”.

The program will address a broad spectrum of fundamental

and applied aspects of these topics.

The meeting provides excellent opportunities to stimulate

scientific discussion and interact with international col-

leagues involved in genome editing, chromosome engi-

neering, advanced cytogenetics and plant breeding.

Support

Organizers

Scientific Committee

Andreas Houben, IPK Gatersleben

Ingo Schubert, IPK Gatersleben

Local Organizing Committee

Regina Devrient, IPK Gatersleben

Katja Koch, IPK Gatersleben

Katrin Menzel, IPK Gatersleben

Sabine Odparlik, IPK Gatersleben

Nicole Wahle, IPK Gatersleben

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Program

Thursday, 30 March 201713:00 Welcome

Chair: Ingo Schubert

13:10 Jaroslav Doležel Institute of Experi-mental Botany, AS CR, Olomouc, Czech Republic

Chromosome genomics supports alien introgression breeding and gene cloning in wheat

13:35 Gabriella Linc Centre for Agricultural Research, Hungarian Academy of Scien-ces, Martonvásár, Hungary

Molecular cytogenetic tools in cha-racterization of pre-breeding mate-rials produced with Agroypron spe-cies

14:00 Petr Capal Institute of Experimental Bo-tany, AS CR, Olomouc, Czech Republic

Single chromosome genomics

14:15 Veit Schubert IPK, Gatersleben

SIM and PALM - two super-resoluti-on methods feasible with the Zeiss Elyra microscope

14:30 coffee break

Chair: Veit Schubert

15:30 Thomas Schmidt Technische Univer-sität Dresden

Comparative Crocus FISHing

15:55 Jiri Macas Biology Centre AS CR, České Budějovice, Czech Republic

Centromere evolution in Fabeae

16:10 Eva Hribova Institute of Experimental Botany, Olomouc, Czech Republic

Comparative analysis of repetitive DNA in eight representatives of fe-scues and ryegrasses

16:25 Phuong Hoang IPK, Gatersleben

Cytogenomics for duckweeds, an emerging crop

16:40 Katrijn Van Laere Institute for Agricul-tural, Fisheries and Food Research, Melle, Belgium

FISH-guided genome assembly in Rosa wichurana

16:55 Lars-Gernot Otto IPK, Gatersleben

Ploidy variation within cultivated Matricaria recutita L. – Towards breeding of sterile triploid chamo-mile

17:10 Michał Kwiatek Institute of Plant Ge-netics of the Polish Academy of Sciences, Poznań, Poland

Constitution and transmission of chromosomes of distant hybrids obtained by intergeneric hybridiza-tions between selected species of goatgrasses (Aegilops spp.) and triticale (×Triticosecale Wittmack)

17:25 Ludmila Khrustaleva (Russian State Agrarian University-Timiryazev Agricultural Academy, Russia

Cytogenetic mapping in Allium and its application for onion bree-ding

17:40 Joanna Lusinska University of Silesia, Katowice, Poland

Analysis of Brachypodium karyoty-pe structure and evolution using cross-species chromosome barco-ding

17: 55 End

19:00 Brewery Quedlinburg

Friday, 31 March 2017

Chair: Jörg Fuchs

9:00 Robert Hasterok University of Silesia, Katowice, Poland

Dissecting grass genome organi-sation at the cytomolecular level using the model genus Brachypo-dium

9:25 Thorben Sprink JKI, Quedlinburg, Germany

Different aspects of Genome Editing in plants using CRISPR/Cas9

9:50 Stefan Hiekel IPK, Gatersleben

Haploid induction after targeted mutagenesis of Cenh3 in barley

10:15 Katharina Unkel JKI, Quedlinburg, Germany

Targeted modifi cations of centro-meric histone H3 (CENH3) by using CRISPR/Cas9 in carrots (Daucus carota L.)

10:30 Takayoshi Ishii IPK, Gatersleben

Dynamics of cowpea CENH3 to-wards haploid induction

10:45 coffee break

Chair: Andreas Houben

11:30 Stefan Heckmann IPK, Gatersleben

Can we harness meiosis for crop plant breeding?

11:55 Nico de Storme Ghent University, Bel-gium

PROTEIN PHOSHATASE 2A pro-tects centromeric sister chromatid cohesion in Arabidopsis male mei-osis I by maintaining REC8 at the chromocenters

12:10 Steven Dreissig IPK, Gatersleben

Single pollen genotyping

12:35 Arita Kuś University of Silesia in Katowi-ce, Poland

Establishing Brachypodium dista-chyon as a model in analysis of plant genomes stability after muta-genic treatment

12:50 Mahmoud Said Institute of Experimen-tal Botany, Olomouc, Czech Republic

The karyotype of Agropyron crista-tum and its comparison with that of bread wheat using FISH with single gene probes

13:05 Mirko Vanetti European Application Manager Functional Genomics

Improved CRISPR genome editing using chemically-modifi ed crRNA:tracrRNA complexes and Cas9 protein

13:20 Lunch break, IPK Casino

14:00 End of meeting

GPZ ‘Cytogenetic’ meeting 2017

Chromosome biology and genome editing in the context of plant breeding

Contact:Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) OT GaterslebenCorrensstraße 3D-06466 Seeland, Germany

Regina DevrientManaging Offi ce | Public RelationsPhone: 0049 039482 5 837Email: gpz2017cg@ipk-gatersleben.de

www.ipk-gatersleben.de/meetings/qpzt-gpz-kvr-2017/

We are looking forward to welcome you in Gatersleben.

Program

30 - 31 March 2017in Gatersleben

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Table of Contents

Topic ..............................................................................3

Support...........................................................................3

Organizers ......................................................................3

Program .....................................................................4

Abstract ..........................................................................7

Chromosome genomics supports alien introgression breeding and gene cloning in wheat

Jaroslav Doležel ........................................................7

Single chromosome genomics

Petr Cápal ..................................................................8

Molecular cytogenetic tools in characterization of pre-breed-ing materials produced with Thinopyrum species

Gabriella Linc .............................................................9

Comparative FISHing of saffron (Crocus sativus L.) and relat-ed Crocus species

Thomas Schmidt ...................................................... 10

Centromere evolution in Fabeae

Jiří Macas ............................................................... 11

FISH-guided genome assembly in Rosa wichurana

Katrijn van Laere ..................................................... 12

Ploidy variation within cultivated Matricaria recutita L. – To-wards breeding of sterile triploid chamomile

Lars-Gernot Otto ..................................................... 13

Constitution and transmission of chromosomes of distant hybrids obtained by intergeneric hybridizations between se-lected species of goatgrasses (Aegilops spp.) and triticale (×Triticosecale Wittmack)

Michał Kwiatek ......................................................... 14

Cytogenetic mapping in Allium and its application for onion breeding.

Ludmila Khrustaleva ................................................ 15

Analysis of Brachypodium karyotype structure and evolution using cross-species chromosome barcoding

Joanna Lusinska ..................................................... 16

Dissecting grass genome organisation at the cytomolecular level using the model Brachypodium

Robert Hasterok ....................................................... 17

Haploid induction after targeted mutagenesis of CENTRO-MERIC HISTONE 3 in barley

Stefan Hiekel ........................................................... 18

Targeted modifications of centromeric histone H3 (CENH3)

by using CRISPR/Cas9 in carrots (Daucus carota L.)

Katharina Unkel ........................................................ 19

PROTEIN PHOSHATASE 2A protects centromeric sister chro-matid cohesion in Arabidopsis male meiosis I by maintaining REC8 at the chromocenters

Nico De Storme ........................................................20

Establishing Brachypodium distachyon as a model in analy-ses of plant genome stability after mutagenic treatment

Arita Kus .................................................................. 21

The karyotype of Agropyron cristatum and its comparison with that of bread wheat using FISH with single gene probes

Mahmoud Said ........................................................22

Increased CRISPR efficiency using chemically-modified and length-optimized crRNA:tracrRNA complexes.

Mirko Vanetti ...........................................................23

List of participants ......................................................... 24

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7

Abstract

Chromosome genomics supports alien introgression breeding and gene cloning in wheatJaroslav Doležel

Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, 78371 Olomouc, Czech Republic e-mail: dolezel@ueb.cas.cz

The analysis of nuclear genomes remains chal-

lenging in many plant species due to genome

complexity and large size. To overcome the diffi-

culties, our team has been developing chromo-

some-centric approaches, which rely on the ability

to dissect nuclear genomes to chromosomes. This

is achieved by preparing suspensions of intact mi-

totic chromosomes from root tip meristems and

isolating chromosomes of interest by flow cytom-

etry. The lossless complexity reduction facilitates

de novo genome assembly as well as validation of

already available whole genome assemblies. For

species where reference genome assemblies are

not yet available, chromosome genomics provides

a cost-effective way to identify a majority of genic

sequences and order them along chromosomes.

Chromosome-derived sequences facilitate devel-

opment of DNA markers to support alien introgres-

sion breeding. With the growing number of fin-

ished reference genome sequences for important

crops, the future of chromosome genomics lies

in the ability to target particular genome regions.

This results in a significant reduction of costs and,

if needed, it allows analyzing a chromosome of in-

terest isolated from different genotypes (mutants).

The applications include identification of chromo-

somes with integrated transgenes, characteriza-

tion of alien chromatin in introgression lines and

development of molecular markers. Gene clon-

ing is becoming one of the most important appli-

cations of chromosome genomics. The targeted

approach greatly streamlines gene cloning and

reduces project costs. Two chromosome-based

gene cloning approaches, namely MutChromSeq

and TACCA (targeted chromosome-based cloning

via long-range assembly) have been developed

and validated recently. Chromosome genomics

can be applied in any species from which a liquid

suspension of intact mitotic chromosomes can be

prepared and the number of uses of flow-sorted

chromosomes in plant genomics keeps growing.

This work has been supported by the National Pro-

gram of Sustainability (grant award LO 2014).

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Single chromosome genomicsPetr Cápal1, Nicolas Blavet1, Jan Vrána1, Takashi R. Endo2, Miroslava Karafiátová1, Jaroslav Doležel1

1 Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, 78371 Olomouc, Czech Republic 2 Faculty of Agriculture, Ryukoku University, 1-5 Yokotani, Seta Oe-cho, Otsu, Shiga 520-2194, Japan

The analysis of complex polyploid and highly

repetetive plant genomes can be significantly sim-

plified by dissecting them into their natural subunits

– chromosomes – by flow cytometry. The sorting

of chromosomes in plants has its limitations due to

their similar size and DNA content. To overcome

this limitation a method for obtaining DNA from

single copies of chromosome was developed.

Each individual copy of a chromosome is 106

amplified to obtain microgram quantities of chro-

mosome-specific DNA that is suitable for various

downstream applications including next-genera-

tion sequencing. Utilizing this approach it is possi-

ble to identify genic sequences on particular chro-

mosomes, to develop chromosome-specific DNA

markers, to verify assignment of DNA sequence

contigs to individual pseudomolecules, and to

validate whole-genome assemblies. The protocol

expands the potential of chromosome genomics,

which may now be applied to any plant species

from which chromosome samples suitable for flow

cytometry can be prepared, and opens new ave-

nues for studies on chromosome structural het-

erozygosity and haplotype phasing in plants.

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Molecular cytogenetic tools in characterization of pre-breeding materials produced with Thinopyrum speciesGabriella Linc* – Eszter Gaal – Diana Icsó – István Molnár - Edina Türkösi

Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Martonvásár 2462, Brunszvik Str.2. Hungary

*Presenting author: Gabriella Linc, e-mail: linc.gabriella@agrar.mta.hu

Wild relatives of cultivated wheat represent a rich

potential source of genetic variation for many ag-

riculturally significant characteristics. Perennial

Triticeae species - genotypes of the Thinopyrum

genus - are important as tertiary gene pools for

wheat improvement. Understanding the organi-

zation of the genomes in the Thinopyrum genus

and their phylogenetic relationships with other re-

lated species will greatly facilitate the utilization of

these species for transferring agronomically useful

genes into bread wheat.

Detailed FISH-based karyotype of three diploid

wheatgrass species, Agropyron cristatum [(L.)

Beauv.] v. Agropyron cristatum (L.) Gaertn., Thi-

nopyrum bessarabicum [(Savul.&Rayss) A. Löve],

Pseudoroegneria spicata [(Pursh) A. Löve], the

supposed ancestors of hexaploid Thinopyrum in-

termedium [(Host) Barkworth & D.R.Dewey] com-

piled using DNA repeats and microsatellite mark-

ers. Fluorescence in situ hybridization (FISH) with

repetitive DNA probes was suitable for the identi-

fication of individual chromosomes of the diploid

JJ, SS and PP genomes. Among seven tested

microsatellite markers only (GAA)n trinucleotide

sequence is appropriate to use as single chromo-

some marker for the Ps. spicata 1S chromosome.

Based on COS marker analysis, phylogenetic re-

lationship between diploid wheatgrasses and the

hexaploid bread wheat genome was established.

One of these findings supports that J and E ge-

nomes are in the neighbouring clusters.

A Thinopyrum intermedium × Thinopyrum pon-

ticum synthetic hybrid wheatgrass is an excellent

source of leaf and stem rust resistance. Pre-breed-

ing materials have been developed in Martonvásár

and wheat line Mv9kr1 was crossed with this hy-

brid (Agropyron glael) in order to transfer its ad-

vantageous agronomic traits into wheat. Progenies

were screened by in situ hybridization and disomic

translocations were selected.

This work was supported by National Science

Foundation Grants OTKA K10855, K104 382;

and the MTA KEP 5/2016 (Hungarian Academy

of Sciences).

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Comparative FISHing of saffron (Crocus sativus L.) and related Crocus species Thomas Schmidt, Gerhard Menzel

Department of Biology, Technische Universität Dresden, 01062 Dresden

The flowers of saffron (Crocus sativus) contain

the most expensive spice of the world, and the

species is grown as crop in rural regions of Spain,

Greece, Iran and Kashmir. Saffron is a triploid hy-

brid (3n = 24, x = 8) with a genome of approxi-

mately 10.5 Gp. Due to its infertility it is only prop-

agated vegetatively, and hence the species shows

only very low genetic variability. The parental spe-

cies of C. sativus are yet not known. Crocus cart-

wrightianus is considered as a donor, however,

also autopolyploidy is discussed.

Several karyotyping experiments have been per-

formed including mostly chromosome staining and

banding. However, due to the lack of discriminat-

ing probes a clear and unequivocal karyotype has

not been established yet. We have performed ge-

nome sequencing of saffron to isolate probes for

FISH. Using RepeatExplorer, we have analysed

the major classes of repetitive sequences includ-

ing many satellite DNA families.

Multi-colour FISH with satellite DNA probes and

rDNA genes generated up to four cytogenetic land-

marks per chromosome resulting in an unequivo-

cal chromosome identification and establishment

of a FISH karyotype. In six of the nine triplets we

found heteromorphic chromosomes strongly indi-

cating the allopolyploid nature of saffron. By inte-

grating FISH signals with reported staining karyo-

types we identified the sequence composition of

large C-banding sites.

Expanding the multi-colour FISH enabled the chro-

mosome identification in seven related Crocus

species. Comparative FISH of the karyotype of C.

cartwrightianus (2n = 16) with saffron chromo-

somes showed many inconsistencies. Although

C. cartwrightianus has most likely contributed to

the chromosome complement of saffron, not all

saffron triplets contain two homologues of C. cart-

wrightianus suggesting that C. cartwrightianus

itself has heteromorphic chromosomes. This is in

line with the variability in the number of satellite

sites found among C. cartwrightianus plants test-

ed.

11

Centromere evolution in FabeaeJiří Macas

Biology Centre CAS, Institute of Plant Molecular Biology, Ceske Budejovice, Czech Republic e-mail: macas@umbr.cas.cz; web: http://w3lamc.umbr.cas.cz/lamc/

The legume tribe Fabeae includes four main genera,

Vicia, Lathyrus, Pisum and Lens, that exhibit extraor-

dinary diversity in the structure and sequence compo-

sition of their centromeres. While Vicia and Lens have

monocentric chromosomes, the primary constrictions

of metaphase chromosomes in Lathyrus and Pisum are

extended up to a third of chromosome length and carry

multiple domains of CenH3-containing chromatin. Since

these constrictions carry histone phosphorylation pat-

terns similar to holocentric chromosomes, it has been

speculated that they represent a transition between

monocentrics and holocentrics. In this talk, I will review

our new data on sequence composition of centromeres

across various Fabeae species differing in centromere

organization. I will also present results of detailed com-

parative analysis of homeologous centromeres between

two Pisum species, P. sativum and P. fulvum, revealing

various mechanisms of their diversification.

12

FISH-guided genome assembly in Rosa wichurana Katrijn van Laere, Ilya Kirov, Ellen de Keyser, Jan de Riek J, Leen Leus, Annelies Haegeman, Chang Liu, Tom Ruttink

The genus Rosa has important economic value in

the ornamental sector and many breeding activi-

ties are going on supported by molecular studies.

To extend the genomic toolbox for rose breeding

we initiated genome sequencing of Rosa wich-

urana, a diploid species involved in the origin of

many modern rose cultivars and a valuable source

of resistance genes. Using Illumina (2x250 reads)

sequencing, Hi-C sequencing and Lachesis, a

genome contact-probability map with 16771 scaf-

folds >10kb ordered into 7 pseudochromosomes

(~0.7 genome equivalent) was built. To validate

this draft genome assembly, high sensitive Tyr-

amide-FISH with 14 single- copy probes along

the 7 Rosa chromosomes have been performed,

revealing good co-linearity between the cytoge-

netic map and the Hi-C based map. In addition

tyramide-FISH with 18 single-copy probes located

on chromosome 7 was done and the order of the

genes was determined. This shows that the long

arm of chromosome 7 is mostly made up of eu-

chromatin, while the short arm consists of heter-

ochromatin, which is very difficult to order prop-

erly by Lachesis. More FISH markers are needed

to come to a good anchoring in this region. FISH

is very valuable to map repetitive regions and to

integrate genome assembly with chromosomal

landmarks, such as heterochromatin and (peri)

centromeric regions, which enables to under-

stand their evolution and function. To determine

the centromere structure and position on the R.

wichurana pseudochromosomes, we identified

rose tandem centromeric repeat sequences in the

repeatome, and visualized those by FISH on mitot-

ic and pachytene chromosomes.

Future work involves the creation of a GBS-based

genetic map and further integration of complemen-

tary cytogenetic, physical and genetic maps. The

resulting high-quality genome assembly, together

with ongoing RNAseq data analysis can be further

applied in rose breeding.

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Ploidy variation within cultivated Matricaria recutita L. – Towards breeding of sterile triploid chamomileLars-Gernot Otto1, Wolfram R. Junghanns2, Andreas Plescher 3, Marlies Sonnenschein3, Bartolome Plocharski3 and Timothy F. Sharbel1

1 Apomixis Research Group, Institute for Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, D-06466 Stadt Seeland OT Gatersleben, Germany; Corresponding author, E-mail: ottolg@web.de 2 Dr. Junghanns GmbH, Aue 182, D-06449 Aschersleben, OT Groß Schierstedt, Germany 3 PHARMAPLANT Arznei- und Gewürzpflanzen Forschungs- und Saatzucht GmbH, Am Westbahnhof 4, D-06556 Artern, Germa-ny

Matricaria recutita L. (German chamomile) has a

long history of medicinal use, being already men-

tioned by Hippocrates (5th century BC). Chamo-

mile is one of the most important medicinal plants

in Germany. New fields for cultivation are difficult

to gain, since the seeds can lay dormant for 10 to

15 years in the soil. Thus, also crop rotation is in-

hibited, leading to the accumulation of chamomile

specific diseases. Cultivated varieties are diploid

or artificially generated tetraploid. A sterile triploid

chamomile variety could be a solution, like in many

fruit and ornamental crop plants for which seeds

are dispensable.

The ploidy variation in various chamomile varieties

and populations was determined by flow-cytome-

try. Several tetraploid varieties contained to some

extent diploid, triploid and aneuploid plants. As a

proof of concept, triploid chamomile plants could

be generated by interploid crosses between di-

and tetraploid parents. The triploid plants were

highly sterile, and of comparable agricultural per-

formance as di- or tetraploid plants.

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Constitution and transmission of chromosomes of distant hybrids obtained by inter-generic hybridizations between selected species of goatgrasses (Aegilops spp.) and triticale (×Triticosecale Wittmack)Michał Kwiatek, Joanna Majka, Maciej Majka, Jolanta Belter, Halina Wiśniewska

Institute of Plant Genetics of the Polish Academy of Sciences; Strzeszyńska 34, 60-479 Poznań, Poland

Spontaneous hybrids arisen from interspecif-

ic and intergeneric crosses are one of the main

parts of evolution. Chromosome rearrangements

are utilized in order to transfer desirable traits into

cultivated plants. The aim of our study was to de-

termine the dynamics of changes in chromosome

constitution of intergeneric hybrids obtained by

crossing hexaploid triticale (×Triticosecale Witt-

mack) with wild Aegilops species. The main as-

sumptions of this study was (1) to precisely identify

and compare the chromosomes of Aegilops spe-

cies and triticale (including relatives from Triticum

genus); (2) to evaluate the localization of chromo-

some breakpoints and (3) to select and study hy-

brid forms bearing valuable traits.

We used five Aegilops spp. × Secale cereale

amphiploid forms for reciprocal distant hybrid-

izations with triticale varieties. We assumed, that

using such forms will have a significant impact on

F1 hybrid stability because of R-genome chromo-

somes, which will be able to pair during prophase

I of meiosis and will ensure the functional daughter

cells formation and sufficient level of vital pollen

grains, as a consequence.

Firstly, we established and compared the FISH

patterns on chromosomes of several Aegilops

and Triticum species and triticale using repetitive

sequences from BAC library of wheat ‘Chinese

Spring’. The differences between localization of

cytogenetic markers in homoeologous chromo-

somes were detected in several species of Ae-

gilops and Triticum genus. The most informative

probes were used for karyotyping of Aegilops-trit-

icale hybrids. Chromosome dynamics was ob-

served in subsequent generations of hybrids

during mitotic metaphase of root meristems and

first metaphase of meiosis of pollen mother cells.

Fluorescence in situ hybridization (FISH) and im-

munolocalization and was applied in order to de-

tect DNA sequences and synaptonemal complex

which are involved in chromosome pairing.

We developed several monosomic/disomic alien

addition/substitudion triticale forms which are

crucial for transfer of genes from wild relatives

into cultivated varieties. Our cytogenetic study,

supported by the marker assisted selection using

Pm13 marker and visual evaluation of infection

by Blumeria graminis, allowed to select triticale

hybrids carrying chromosome 3Sv (derived from

Ae. variabilis) which were tolerant to the powdery

mildew. We allocated chromosome 2D of Ae.

tauschii in triticale background, which resulted

in changes of its organization, what was related

to varied expression of agronomically important

traits. Moreover, we investigated the least known

gametocidal action of 4Mg chromosome (derived

from Ae. geniculata) during the meiosis of pollen

mother cells of monosomic 4Mg addition triticale

plants. We adapted the gametocidal system in

purpose to induce the chromosome aberrations

between Triticum and Secale chromosomes of

triticale. We applied this mechanism in a combi-

nation with DH lines production, which provided a

sufficiently large population of homozygous dou-

bled haploid individuals of triticale with two identi-

cal copies of translocation chromosomes.

The study was supported by a grant from the

National Sciences Centre (NCN SONATA 6;

2013/11/D/NZ9/02719).

15

Cytogenetic mapping in Allium and its application for onion breeding.Ludmila Khrustaleva1 , Dmitry Romanov1, Ilya Kirov1, Jiming Jiang2, and Michael J. Havey3

1 Center of Molecular Biotechnology, Department of Genetics, Biotechnology Plant Breeding and Seed Science, Rus-sian State Agrarian University-Timiryazev Agricultural Academy, 49, Timiryazevskaya Str., 127550 Moscow, Russia; 2 Department of Horticulture, University of Wisconsin, Madison, WI 53706 USA;3 USDA-ARS and Department of Hor-ticulture, University of Wisconsin, Madison, WI 53706 USA

Fluorescence in situ hybridization (FISH) has not

been readily exploited for physical mapping of

molecular markers in plants due to the technical

challenge of visualizing small single-copy probes.

Signal amplification using tyramide FISH can in-

crease sensitivity up to 100 fold. We have applied

tyramide-FISH for visualization of gene/marker on

the Allium chromosomes.

Tyramide FISH was used to locate relatively small

genomic amplicons (846 to 2251 bp) and a cDNA

clone (666 bp) from molecular markers linked to

Ms locus onto onion chromosome 2 near the cen-

tromere, a region of relatively low recombination.

This result explains why several labs have identified

molecular markers tightly linked to the Ms locus af-

ter screening relatively few DNA clones or primers

and segregating progenies. Although these mark-

ers are still useful for marker-aided selection, our

results indicate that map-based cloning of Ms will

likely be difficult due to reduced recombination near

this gene.

Onion chromosome 5 carries major quantitative

trait loci (QTL) of interest to breeders that control

dry-matter content, pungency and storability of

bulbs etc. We used EST clones and sequences

of onion from the NCBI database to develop DNA

probes for in situ hybridization. We produced DNA

probes that carried introns to increase the hybrid-

ization specificity of the probes. Through the inte-

gration of genetic and chromosomal maps we were

able to estimate the distribution of recombination

events along onion chromosome 5.

We cloned, sequenced and located the alliinase

(probe 1100bp) and lacrymatory factor synthase

(LFS, probe 550bp) genes that encode enzymes

operating in a biochemical pathway that produces

the compounds responsible for the onion’s charac-

teristic flavour. A disruption of collinearity between

homeologous chromosomes was revealed by map-

ping the alliinase genes in a number of Alliium spe-

cies closely related to onion. This information can

be useful for effective interspecific breeding be-

cause genome collinearity is a strong prerequisite

for homologous recombination and transferring de-

sirable traits from donor species.

This study was financially partly supported by a

research grant № 16-16-10031 from the Russian

Science Foundation.

16

Analysis of Brachypodium karyotype structure and evolution using cross-species chromosome barcodingJoanna Lusinska, Elzbieta Wolny, Robert Hasterok

Department of Plant Anatomy and Cytology, Faculty of Biology and Environmental Protection, University of Silesia in Katowice, 28 Jagiellonska Street, 40–032 Katowice, Poland, e-mail: jlusinska@us.edu.pl

The enormous diversity of angiosperm plants is a

reflection of great variation in their genomes. Re-

cent data indicate that polyploidisation and dys-

ploidy are the major evolutionary forces driving the

success of flowering plants and also the most im-

portant mechanisms, which determine a numerical

alteration of chromosomes. Paleogenomic studies

based on the bioinformatics analyses of DNA se-

quences have revealed that nested chromosome

fusions played an important role in the divergence

of modern grasses.

The genus Brachypodium represents a particular-

ly suitable model system for the analysis of grass

karyotype evolution. It comprises 15–20 species

with different basic chromosome numbers, size,

morphology and ploidy levels. Present study elu-

cidate the mechanisms of the chromosome re-

arrangements that have shaped the structure of

Brachypodium karyotypes, using comparative

chromosome barcoding by BAC-FISH (fluores-

cence in situ hybridisation with bacterial artificial

chromosomes as probes). The karyotypes of se-

lected Brachypodium species were compared

with reference to the model grass B. distachyon.

Single-locus BAC clones derived from the B. dis-

tachyon genomic libraries were selected from

the assemblies of FPCs (FingerPrinted Contigs)

that had previously been assigned to the chro-

mosomes of B. distachyon. This comparative

chromosome barcoding approach can be used to

study the organisation of karyotypes and recon-

struct mechanisms of the chromosome rearrange-

ments that have shaped the structure of the extant

grasses karyotypes.

This work is supported by the National Sci-

ence Centre Poland (grant no. 2012/04/A/

NZ3/00572).

17

Dissecting grass genome organisation at the cytomolecular level using the model Brachypodium Robert Hasterok, Alexander Betekhtin, Natalia Borowska-Zuchowska, Agnieszka Braszewska-Zalewska, Karolina Chwialkowska*, Marta Hosiawa-Baranska, Dominika Idziak-Helmcke, Arita Kus, Jolanta Kwasniewska, Miroslaw Kwasniewski*, Joanna Lusinska, Ewa Robaszkiewicz, Magdalena Rojek, Rakesh Sinha, Alexandra Skalska, Marta Sowa, Elzbieta Wolny, Karolina Zubrzycka,

Department of Plant Anatomy and Cytology / *Department of Genetics, Faculty of Biology and Environmental Protection, Universi-ty of Silesia in Katowice, 28 Jagiellonska Street, 40–032 Katowice, Poland, e-mail: robert.hasterok@us.edu.pl

Modern molecular cytogenetics combines various

methodological approaches of cytology, molecu-

lar genetics and advanced digital image analysis.

It focuses on the study of nuclear genomes at the

microscopic level. The cytomolecular organisation

of plant genomes is still rather poorly investigated,

compared to that of animals. Most plant genomes,

including those of economically and ecologically

crucial cereals and forage grasses, are usually

large and saturated with repetitive DNA, which

hampers detailed molecular cytogenetic analyses.

Model organisms possess a combination of fea-

tures, which makes them more amenable to sci-

entific investigation than others. One of the most

recent and rapidly developing model systems are

representatives of the Brachypodium genus, par-

ticularly B. distachyon. They possess small, and

in some cases, already sequenced genomes with

a low repeat content, diverse basic chromosome

numbers and ploidy levels. They also have an in-

teresting phylogeny, short life cycles and simple

growth requirements, complemented by a rapidly

and continuously growing repertoire of various ex-

perimental tools.

This presentation outlines and discusses our cur-

rent projects and their future prospects, using

Brachypodium species for research on various

aspects of grass genome organisation, e.g. (i)

karyotype structure and evolution, (ii) distribution

of chromosome territories within the nucleus, (iii)

dynamics of epigenetic modifications of chromatin

during embryo development and cell differentia-

tion, (iv) true nature of selective silencing of rRNA

genes in some Brachypodium allopolyploids and

(v) instability of a small grass genome induced via

mutagenic treatments.

This work is supported by the Nation-

al Science Centre Poland (grants no.

2012/04/A/NZ3/00572, 2014/14/M/

NZ2/00519 and 2015/18/M/NZ2/00394).

18

Haploid induction after targeted mutagenesis of CENTROMERIC HISTONE 3 in barleyStefan Hiekel, Maia Gurushidze, Sindy Schedel, Takayoshi Ishii, Andreas Houben and Jochen Kumlehn

Many plant breeding programs rely on the gen-

eration of homozygous lines after cross-combi-

nation of parental plants with different desired

traits. Genetically stable lines can be produced

either by time-consuming and laborious selfing

over numerous generations or in just one step by

employing haploid technology. Likewise, methods

of doubled haploid production can also greatly

facilitate the generation of homozygous experi-

mental recombinants, induced mutants as well as

transgenic and genome engineered plants. Due to

various constraints of current haploid technology

(e.g. genotype-dependency, challenging cell cul-

ture procedures, recombination bias in DH-pop-

ulations) there is a strong demand for alternative

or even universally useful methods applicable

in many crop species. Therefore, we aim to es-

tablish a novel method in the model cereal crop

barley based upon uni-parental genome elimina-

tion as a result of a functional modification in the

centromere-specific histone 3 (CENH3) - a prin-

ciple recently discovered in Arabidopsis. CENH3

replaces canonical HISTONE 3 in centromeric

nucleosomes in many eukaryotic species. The

CENH3 protein recruits key components of the

kinetochore complex and is therefore essential

for proper chromosome segregation during mei-

otic and mitotic cell divisions. In Arabidopsis,

maize and more recently in Brassica juncea, the

replacement of native CENH3 by an altered deriv-

ative (GFP-tailswap-CENH3) was demonstrated to

result in plants having a certain capacity of pro-

ducing haploid progeny. Crossed with any CENH3

wild type plant of the same species, these lines

trigger the elimination of their own chromosomes

during early embryo development (Kelliher et al.,

2016; Ravi and Chan, 2010; Watts et al., 2017).

To produce such inducer-lines for barley, we sta-

bly expressed a GFP-tailswap-HvCENH3α trans-

gene and confirmed the localization of its product

to all barley centromeres. In addition, we created

functional knock out (KO) alleles of HvCENH3№

via targeted mutagenesis using RNA-guided en-

donucleases (RGENs). Plants carrying a cenh3№

KO allele in homozygous condition exhibit a nor-

mal growth phenotype but do not produce any

progeny upon self-pollination. However, crossing

of these mutants with wild type barley results in

the elimination of the cenh3№ KO allele-carrying

genome, which, via embryo rescue, can entail

the formation of haploid plants. Surprisingly, the

GFP-tailswap-HvCENH3α is neither essential for

the induction of haploidy nor does it rescue the

infertility of HvCENH3α loss-of-function mutants.

Literature:

Kelliher, T., Starr, D., Wang, W., McCuiston, J.,

Zhong, H., Nuccio, M.L., and Martin, B. 2016.

Maternal haploids are preferentially induced by

CENH3-tailswap transgenic complementation in

maize. Frontiers in Plant Science 7.

Ravi, M. and Chan, S.W. 2010. Haploid plants

produced by centromere-mediated genome elim-

ination. Nature 464:615-618.

Watts, A., Singh, S.K., Bhadouria, J., Naresh, V.,

Bishoyi, A.K., Geetha, K.A., Chamola, R., Pattan-

ayak, D., and Bhat, S.R. 2017. Brassica juncea

Lines with Substituted Chimeric GFP-CENH3 Give

Haploid and Aneuploid Progenies on Crossing

with Other Lines. Frontiers in Plant Science 7.

19

Targeted modifications of centromeric histone H3 (CENH3) by using CRISPR/Cas9 in carrots (Daucus carota L.)Katharina Unkel1, Thorben Sprink2, Holger Budahn1, Frank Dunemann1

1Julius Kühn - Institut (JKI), Federal Research Center for Cultivated Plants, Institute for Breeding Research on Horti-cultural Crops, Erwin-Baur-Str. 27, 06484 Quedlinburg, Germany

2Julius Kühn - Institut (JKI), Federal Research Center for Cultivated Plants, Institute for Biosafety in Plant Biotechnol-ogy, Erwin-Baur-Str. 27, 06484 Quedlinburg, Germany

Carrot (Daucus carota L.) is the most widely grown spe-

cies of the genus Daucus, with F1 hybrid breeding as

the main breeding technique. However, as a cross-pol-

linated biannual species, the production of genetically

homogeneous parental lines through several subse-

quent steps of inbreeding is long lasting and might lead

to inbreeding depression. Haploid production by tissue

culture techniques is inefficient in Apiaceae species,

and genome elimination by interspecific hybridization

has not yet been reported for the Daucus genus. Modi-

fication of the kinetochore specific centromeric histone

H3 (CENH3) - which plays a major part in proper segre-

gation of chromosomes during cell division - might result

in uniparental genome elimination during early embryo-

genesis and has been proposed as a new accelerat-

ed method for haploid induction. In eudicots CENH3

consists of a highly conserved C-terminal Histone Fold

Domain (HFD) and an N-terminal tail showing variations

in length and sequence between species. We used the

RNA guided endonuclease (RGEN) technique CRISPR/

Cas9 to induce mutations at different sites of the HFD

region of DcCENH3 with the objective to generate se-

quence variants which impair the function of CENH3

in the light of creating potential haploid inducer lines.

To complement the possible loss of function of carrot

CENH3, we additionally co-transformed carrot varieties

with the PgCENH3 gene cloned from Panax ginseng,

a member of the Araliaceae plant family belonging to

the order Apiales. Due to the high regeneration rate of

hairy roots, an early screening method to identify the

most promising hairy root lines is essential prior to plant

regeneration via somatic embryogenesis. Among oth-

er mutation detection techniques, we used high-reso-

lution fragment (HRF) analysis via an automatic LICOR

sequencing apparatus as a pre-selection tool to identify

multiple CRISPR/Cas9 induced mutations inside the

coding sequence of DcCENH3. We show that we were

able to induce mutations in the CENH3 gene of carrot

by RGEN which led to visible changes in the CENH3

phenotype in some hairy root lines.

20

PROTEIN PHOSHATASE 2A protects centromeric sister chromatid cohesion in Arabi-dopsis male meiosis I by maintaining REC8 at the chromocentersGuoliang Yuan, Nico De Storme, Arp Schnittger, Cathrine Lillo and Danny Geelen

During meiosis, the cohesin complex that maintains

sister chromatid cohesion is lost in a stepwise manner.

In yeast and vertebrates, the meiosis-specific cohesin

subunit Rec8 is cleaved only along the chromosome

arms at meiosis I; up till Metaphase II it is protected at

the centromeres by the action of Shugoshin (Sgo) and

Protein Phosphatase 2A (PP2A). In plants, centromeric

sister chromatid cohesion from Metaphase I to II is pro-

tected by two Sgo orthologs and by a plant-specific pro-

tein PATRONUS (PANS), however the detailed mecha-

nism by which sister chromatid cohesion is maintained

at centromeres is still poorly understood. The Arabidop-

sis genome contains nine PP2A B’ subunit homologs.

Using genetic studies we here demonstrate that pp2a b’

№ pp2a b’ α double mutants display premature separation

of sister chromatids in meiosis starting from anaphase I,

whereas single mutants do not show any alteration in co-

hesion release, indicating that AtPP2A B’ α and AtPP2A

B’ α are redundantly required for the maintenance of

centromeric sister chromatid cohesion during meiosis I.

Furthermore, we demonstrate that the AtREC8 cohesin

subunit is prematurely depleted from the centromeric

regions in male meiocytes of the pp2a b’ № pp2a b’ α

double mutant, suggesting that PP2A maintains centro-

meric sister chromatid cohesion from Metaphase I to

II by protecting REC8 from cleavage. Finally, we also

found that AtPP2A B’ α and α are dispensable for mitotic

cell progression in Arabidopsis.

21

Establishing Brachypodium distachyon as a model in analyses of plant genome stabi-lity after mutagenic treatment Arita Kus, Jolanta Kwasniewska, Robert Hasterok

Department of Plant Anatomy and Cytology, Faculty of Biology and Environmental Protection, University of Silesia in Katowice, 28 Jagiellonska Street, 40–032 Katowice, Poland, e-mail: arkus@us.edu.pl

Due to their high sensitivity, higher plants are

widely used for screening and monitoring environ-

mental genotoxicity. Brachypodium distachyon,

an internationally accepted model grass species,

would be a convenient system in mutagenesis to

analyse ‘hot spots’ (and ’cold spots’) of DNA dam-

age in nuclear genome and consequently could

find practical application in the environmental

monitoring. The chromosome rearrangements

are commonly identified using classical cytoge-

netic techniques. Physical mapping technology

together with the availability of BAC libraries of B.

distachyon nuclear DNA, allow comprehensive

analyses of mutagenic effects at the chromosomal

level and extend our understanding of the mech-

anisms of chromosomal aberrations. The visual-

isation of mutagen-induced genome changes,

including micronuclei formation and alterations of

chromosome territories in interphase nuclei using

fluorescence in situ hybridisation (FISH) with se-

lected chromosome-specific BAC clones, as well

as ribosomal DNA and chromosome region-spe-

cific, i.e. centromeric and telomeric probes here

is presented.

This work is supported by the National Sci-

ence Centre Poland (grant no. 2012/04/A/

NZ3/00572).

22

The karyotype of Agropyron cristatum and its comparison with that of bread wheat using FISH with single gene probesMahmoud Said1, Tatiana Danilova2, Eva Hřibová1, Bernd Friebe2, Bikram S. Gill2, Jan Vrána1, Jaroslav Doležel1

1Institute of Experimental Botany AS CR, Olomouc, Czech Republic 2Wheat Genetic Resources Center, Kansas State University, Manhattan, KS, USA

e-mail: said@ueb.cas.cz

Agropyron cristatum L. (2n=2x=14, PP) common-

ly known as crested wheatgrass is a wild relative of

wheat and an attractive source of novel genes for

its improvement. As alien gene transfer by inter-

specific hybridization is affected by chromosome

colinearity, it is important to establish the synten-

ic relationships between the chromosomes of the

donor alien species and wheat. To date, identifi-

cation of all chromosomes of A. cristatum is not

possible and its molecular karyotype has not been

developed. With the aim to identify chromosomes

of A. cristatum by FISH, its genomic DNA was se-

quenced and several tandem repeats were discov-

ered. Their location on mitotic chromosomes by

FISH revealed specific distribution pattern for six of

them. The use of one tandem repeat together with

45S rDNA as probes for FISH enabled identifica-

tion of all seven chromosomes of A. cristatum. In

order to analyze the structure and homoeology of

A. cristatum chromosomes, 45 FLcDNA from the

seven chromosome groups of wheat were local-

ized by FISH on chromosomes of crested wheat-

grass cv. Parkway. Important structural rearrange-

ments were observed for chromosomes 2P, 4P,

6P and 7P, while, no major rearrangements were

detected for the remaining three chromosomes.

The results of this work provide new insights into

the genome evolution within the tribe Triticeae and

will facilitate the use of crested wheatgrass in alien

introgression breeding of bread wheat.

23

Increased CRISPR efficiency using chemically-modified and length-optimized crR-NA:tracrRNA complexes.Ashley M. Jacobi, Michael A. Collingwood, Mollie S. Schubert, Garrett R. Rettig, and Mark A. Behlke

Integrated DNA Technologies, Inc. Coralville, IA, 52241 USA

The natural CRISPR-Cas9 system in S. pyogenes

employs two RNA molecules, a 42-nt target-spe-

cific CRISPR RNA (crRNA) and an 89-nt univer-

sal trans-activating RNA (tracrRNA). Through

systematic testing of truncations in the RNAs, we

developed length-optimized versions that exhibit

improved editing performance in mammalian cells.

Although unmodified RNA oligonucleotides can

be used to direct Cas9 cleavage, they are rapidly

degraded by serum or cellular nucleases, limiting

their functional activity. Further, unmodified RNAs

can trigger an innate immune response in mamma-

lian cells. Extensive studies of chemical modifica-

tion strategies for both the crRNA and the tracrR-

NA were performed. Over 400 RNA oligos were

compared for functional performance in various

settings, systematically evaluating the tolerance

of each base for modification. Highly functional

modified variants were developed where as high

as 78% of the crRNA and 84% of the tracrRNA

residues were substituted with 2’OMe RNA. Use

of phosphorothioate modified internucleotide link-

ages or other end-blocking strategies were also

helpful in preventing 5’- or 3’-exonuclease attack.

The new chemically-modified crRNA:tracrRNA

synthetic oligonucleotides can be annealed, com-

plexed with recombinant Cas9 protein and intro-

duced into mammalian cells using lipofection or

electroporation to achieve high editing efficiency

with minimal side effects. Functional validation has

been obtained in a variety of systems including:

mice, zebrafish, nematodes, mammalian tissue

culture cells, iPSCs, and primary T-cells isolated

from human donors.

24

List of participants(Registration till 22. February 2017)

Avila Robledillo, Laura, Biology Centre CAS robledillo@umbr.cas.czAyoub, Mohammad, IPK Gatersleben mohammad.ayoub@agr.cu.edu.egBartoš, Jan, Institute of Experimental Botany bartos@ueb.cas.czBudhagatapalli, Nagaveni, Dr. IPK Gatersleben budhagatapalli@ipk-gatersleben.deCapal, Petr, PhD IEB Olomouc capal@ueb.cas.czCuacos, Maria, Dr. IPK Gatersleben cuacos@ipk-gatersleben.deDe Storme, Nico, Ir University of Ghent nico.destorme@ugent.beDemidov, Dmitri, IPK Gatersleben demidov@ipk-gatersleben.deDoležel, Jaroslav, Prof Ing Institute of Experimental Botany AS CR dolezel@ueb.cas.czDreissig, Steven, IPK Gatersleben dreissig@ipk-gatersleben.deDunemann, Frank, Dr. Julius-Kühn-Institut frank.dunemann@julius-kuehn.deFuchs, Joerg, Dr. IPK Gatersleben fuchs@ipk-gatersleben.deGnad, Heike, Dr. Saaten-Union Biotec GmbH gnad@saaten-union-biotec.comHalbach, Thomas, Dr. Strube Research GmbH & Co. KG t.halbach@strube-research.netHartung, Frank, JKI Quedlinburg frank.hartung@jki.bund.deHasterok, Robert, Prof University of Silesia in Katowice robert.hasterok@us.edu.plHeckmann, Stefan, Dr. IPK Gatersleben heckmann@ipk-gatersleben.deHensel, Götz, Dr. IPK Gatersleben hensel@ipk-gatersleben.deHertig, Christian, IPK Gatersleben hertig@ipk-gatersleben.deHiekel, Stefan, IPK Gatersleben hiekel@ipk-gatersleben.deHoang, Thi Nhu Phuong, IPK Gatersleben hoangt@ipk-gatersleben.deHoffi e, Robert Eric, IPK Gatersleben hoffi er@ipk-gatersleben.deHouben, Andreas, Dr. IPK Gatersleben houben@ipk-gatersleben.deHribova, Eva, PhD Institute of Experimental Botany hribova@ueb.cas.czIshii, Takayoshi, IPK Gatersleben ishii@ipk-gatersleben.deKarafi átová, Miroslava, Dr. lEB As cR karafi atova@ueb.cas.czKempe, Katja, Strube Research GmbH & Co. KG k.kempe@strube-research.netKhrustaleva, Ludmila, Russian State Agrarian University ludmila.khrustaleva19@gmail.comKloiber-Maitz, Monika, Dr. KWS SAAT SE monika.kloiber-maitz@kws.comKumlehn, Jochen, Dr. IPK Gatersleben kumlehn@ipk-gatersleben.deKuś, Arita, University of Silesia in Katowice arkus@us.edu.plKwiatek, Michał, Dr. Institute of Plant Genetics of the Polish Academy of Sciences mkwi@igr.poznan.plLermontova, Inna, Dr. IPK Gatersleben lermonto@ipk-gatersleben.deLinc, Gabriella, Ph.D. Hungarian Academy of Sciences linc.gabriella@agrar.mta.huLührs, Renate, Dr. Saaten-Union Biotec GmbH luehrs@saaten-union-biotec.comŁusińska, Johanna, University of Silesia in Katowice jlusinska@us.edu.plLysak,, Martin, Assoc. Prof. Mgr. Masaryk University martin.lysak@ceitec.muni.czMacas, Jiri, PhD Institute of Plant Molecular Biology macas@umbr.cas.czMandakova,, Terezie, Ph.D. Masaryk University terezie.mandakova@ceitec.muni.czMenzel, Gerhard, Dr. Technische Universität Dresden gerhard.menzel@tu-dresden.deMunicio Diaz, Celia, IPK Gatersleben municio@ipk-gatersleben.deNěmečková, Alžběta, Institute of Experimental Botany nemeckova@ueb.cas.czNeu, Nina, LUH Hannover ninaneu@gmx.de

Schulz, Martin, Dr. genius gmbh martin.schulz@genius.deSpiller, Monika, Dr. Syngenta Seeds GmbH monika.spiller@syngenta.comSteckenborn, Stefan, IPK Gatersleben coria@ipk-gatersleben.deStöver, Anita, Klemm + Sohn GmbH & Co. KG a.stoever@selecta-one.comUnkel, Katharina, Julius-Kühn-Institut katharina.unkel@julius-kuehn.deVan Laere, Katrijn, Dr.ir. Flanders research institute for Agricultural, Fisheries and Food katrijn.vanlaere@ilvo.vlaanderen.be

Vanetti, Mirko, Dr. Integrated DNA Technologies Germany GmbH mvanetti@idtdna.comWeyen, Jens, Dr. Saatzucht Josef Breun GmbH & Co. KG weyen@breun.deWhitbread, Amy, Karlsruhe Institute of Technology amy.whitbread@kit.eduZelkowski, Mateusz, IPK Gatersleben zelkowski@ipk-gatersleben.de

Pouch, Milan, Masaryk University milan.pouch@ceitec.muni.czRuban, Alevtina, IPK Gatersleben ruban@ipk-gatersleben.deSaid, Mahmoud, PhD Institute of Experimental Botany AS CR Said@ueb.cas.czSandmann, Michael, IPK Gatersleben sandmann@ipk-gatersleben.deSchedel, Sindy, IPK Gatersleben schedel@ipk-gatersleben.deSchmidt, Thomas, Prof. Dr. Technische Universität Dresden thomas.schmidt@tu-dresden.deSchnepel, Ines, Klemm + Sohn GmbH & Co. KG i.schnepel@selecta-one.comSchubert, Ingo, Prof. IPK Gatersleben schubert@ipk-gatersleben.deSchubert, Veit, PD Dr. IPK Gatersleben schubertv@ipk-gatersleben.de

Otto, Lars-Gernot, Dr. IPK Gatersleben ottol@ipk-gatersleben.dePlath, Susann, Julius-Kühn-Institut susann.plath@julius-kuehn.de

25

Schulz, Martin, Dr. genius gmbh martin.schulz@genius.deSpiller, Monika, Dr. Syngenta Seeds GmbH monika.spiller@syngenta.comSteckenborn, Stefan, IPK Gatersleben coria@ipk-gatersleben.deStöver, Anita, Klemm + Sohn GmbH & Co. KG a.stoever@selecta-one.comUnkel, Katharina, Julius-Kühn-Institut katharina.unkel@julius-kuehn.deVan Laere, Katrijn, Dr.ir. Flanders research institute for Agricultural, Fisheries and Food katrijn.vanlaere@ilvo.vlaanderen.be

Vanetti, Mirko, Dr. Integrated DNA Technologies Germany GmbH mvanetti@idtdna.comWeyen, Jens, Dr. Saatzucht Josef Breun GmbH & Co. KG weyen@breun.deWhitbread, Amy, Karlsruhe Institute of Technology amy.whitbread@kit.eduZelkowski, Mateusz, IPK Gatersleben zelkowski@ipk-gatersleben.de

Pouch, Milan, Masaryk University milan.pouch@ceitec.muni.czRuban, Alevtina, IPK Gatersleben ruban@ipk-gatersleben.deSaid, Mahmoud, PhD Institute of Experimental Botany AS CR Said@ueb.cas.czSandmann, Michael, IPK Gatersleben sandmann@ipk-gatersleben.deSchedel, Sindy, IPK Gatersleben schedel@ipk-gatersleben.deSchmidt, Thomas, Prof. Dr. Technische Universität Dresden thomas.schmidt@tu-dresden.deSchnepel, Ines, Klemm + Sohn GmbH & Co. KG i.schnepel@selecta-one.comSchubert, Ingo, Prof. IPK Gatersleben schubert@ipk-gatersleben.deSchubert, Veit, PD Dr. IPK Gatersleben schubertv@ipk-gatersleben.de

Otto, Lars-Gernot, Dr. IPK Gatersleben ottol@ipk-gatersleben.dePlath, Susann, Julius-Kühn-Institut susann.plath@julius-kuehn.de

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